Methylglyoxal and a spinal TRPA1-AC1-Epac cascade facilitate pain in the db/db mouse model of type 2 diabetes.

Painful diabetic neuropathy (PDN) is a devastating neurological complication of diabetes. Methylglyoxal (MG) is a reactive metabolite whose elevation in the plasma corresponds to PDN in patients and pain-like behavior in rodent models of type 1 and type 2 diabetes. Here, we addressed the MG-related spinal mechanisms of PDN in type 2 diabetes using db/db mice, an established model of type 2 diabetes, and intrathecal injection of MG in conventional C57BL/6J mice. Administration of either a MG scavenger (GERP10) or a vector overexpressing glyoxalase 1, the catabolic enzyme for MG, attenuated heat hypersensitivity in db/db mice. In C57BL/6J mice, intrathecal administration of MG produced signs of both evoked (heat and mechanical hypersensitivity) and affective (conditioned place avoidance) pain. MG-induced Ca2+ mobilization in lamina II dorsal horn neurons of C57BL/6J mice was exacerbated in db/db, suggestive of MG-evoked central sensitization. Pharmacological and/or genetic inhibition of transient receptor potential ankyrin subtype 1 (TRPA1), adenylyl cyclase type 1 (AC1), protein kinase A (PKA), or exchange protein directly activated by cyclic adenosine monophosphate (Epac) blocked MG-evoked hypersensitivity in C57BL/6J mice. Similarly, intrathecal administration of GERP10, or inhibitors of TRPA1 (HC030031), AC1 (NB001), or Epac (HJC-0197) attenuated hypersensitivity in db/db mice. We conclude that MG and sensitization of a spinal TRPA1-AC1-Epac signaling cascade facilitate PDN in db/db mice. Our results warrant clinical investigation of MG scavengers, glyoxalase inducers, and spinally-directed pharmacological inhibitors of a MG-TRPA1-AC1-Epac pathway for the treatment of PDN in type 2 diabetes.

An evaluative tool for rapid assessment of derelict vessel effects on coastal resources.

Derelict vessels impact coastal and estuarine habitats, fisheries resources, are aesthetically unappealing, and may be a hazard to navigation and recreation. The Government Accountability Office estimated in 2013 over 5600 derelict vessels existed throughout the coastal United States. Considering the large number of derelict vessels present in coastal areas, effective tools are needed to assess the environmental damage exerted by derelict vessels and aid in management strategies for their removal. After carefully reviewing regulations, we developed a 100-point scoring rubric (DVET) to evaluate damage by derelict vessels to natural resources with minimal field effort. The DVET's ability to rapidly assess a derelict vessel's impact on surrounding natural resources was confirmed with additional rigorous sampling and suggest environmental enhancement following vessel removal. The DVET shows promise for informing derelict vessel removal strategies, although more work is needed to quantify environmental benefits of derelict vessel removal and establish guidelines for removal prioritization.

Vagus nerve stimulation and heart rate variability: A scoping review of a somatic oscillatory signal.

The goal of this review is to synthesize the literature on vagus nerve stimulator (VNS)-related changes in heart rate variability (HRV) in patients with drug-resistant epilepsy (DRE) and assess the role of these changes in seizure relief. A scoping literature review was performed with the following inclusion criteria: primary articles written in English, involved implantable VNS in humans, and had HRV as a primary outcome. Twenty-nine studies were retrieved, however with considerable heterogeneity in study methods. The overall depression in HRV seen in DRE patients compared to healthy controls persisted even after VNS implant, indicating that achieving "healthy" HRV is not necessary for VNS therapeutic success. Within DRE patients, changes in frequency domain parameters six months after VNS implant returned to baseline after a year. The mechanism of how VNS reduces seizure burden does not appear to be significantly related to alterations in baseline HRV. However, the subtlety of sympathetic/parasympathetic signaling likely requires a more structured approach to experimental and analytic techniques than currently found in the literature.

Efficacy of vagus nerve stimulation in managing drug-resistant absence epilepsy syndromes.

PURPOSE: Around 11% of patients with absence epilepsy develop drug-resistant absence epilepsy (DRAE), and are at increased risk for developing psychiatric and neurologic comorbidities. Current therapeutic options for DRAE are limited. The purpose of this study was to assess the efficacy of vagus nerve stimulation (VNS) in treating DRAE. METHODS: Our institution maintains a database of patients who received VNS between 2010 and 2022. We identified DRAE patients who were <18 years of age at seizure onset, were electro-clinically diagnosed with an absence epilepsy syndrome (childhood absence, juvenile absence, or Jeavons Syndrome) by an epileptologist, and had normal brain imaging. The primary outcome measure was post-VNS absence seizure frequency. RESULTS: Twenty-six patients (M/F:14/12) were identified. Median age at seizure onset was 7 years (IQR 4-10) and patients experienced seizures for 6 years (IQR 4.3-7.6) before VNS. After VNS, the median absence seizure frequency reduced to 1.5 days (IQR 0.1-3.5) per week from 7 days (IQR 7-7), a 66% reduction seizure frequency. VNS responder rate was 80%, and seven patients achieved seizure freedom. There was no significant effect on VNS efficacy between the time from DRAE diagnosis to VNS placement (p = 0.067) nor the time from first seizure onset to VNS implant (p = 0.80). The median follow-up duration was 4.1 years (IQR 2.4-6.7), without any significant association between follow-up duration and VNS efficacy (r2=0.023) CONCLUSIONS: VNS is effective in managing DRAE. The responder rate was 80%; seizure improvement was independent of age at both seizure onset and latency to VNS after meeting DRAE criteria.

Economic impacts of marine debris encounters on commercial shrimping.

The commercial shrimping industry is subjected to myriad stressors that have led to financial hardships among industry members. One of these stressors is marine debris; however, there is limited understanding of the type and magnitude of impacts. Quantitative methods of estimating the economic impacts of marine debris on the commercial shrimping industry were developed. From June to December 2019, participating shrimpers submitted 393 daily summaries, including shrimping activities, marine debris encounters, damages, and impacts. The impacts of marine debris encounters were assessed from reports of daily damages to fishing assets, daily lost fishing time, daily loss-catch ratios, and daily catch losses. The results of this study demonstrate substantial negative economic impacts on commercial shrimpers related to marine debris encounters. About 17 % of shrimp caught were lost due to marine debris encounters, resulting in foregone total sales and job impacts of $3.2 million and 33 jobs in shrimping and associated businesses.

A hypothesis: Potential contributions of metals to the pathogenesis of pulmonary artery hypertension.

Pulmonary artery hypertension (PAH) is characterized by vasoconstriction and vascular remodeling resulting in both increased pulmonary vascular resistance (PVR) and pulmonary artery pressure (PAP). The chronic and high-pressure stress experienced by endothelial cells can give rise to inflammation, oxidative stress, and infiltration by immune cells. However, there is no clearly defined mechanism for PAH and available treatment options only provide limited symptomatic relief. Due to the far-reaching effects of metal exposures, the interaction between metals and the pulmonary vasculature is of particular interest. This review will briefly introduce the pathophysiology of PAH and then focus on the potential roles of metals, including essential and non-essential metals in the pathogenic process in the pulmonary arteries and right heart, which may be linked to PAH. Based on available data from human studies of occupational or environmental metal exposure, including lead, antimony, iron, and copper, the hypothesis of metals contributing to the pathogenesis of PAH is proposed as potential risk factors and underlying mechanisms for PAH. We propose that metals may initiate or exacerbate the pathogenesis of PAH, by providing potential mechanism by which metals interact with hypoxia-inducible factor and tumor suppressor p53 to modulate their downstream cellular proliferation pathways. These need further investigation. Additionally, we present future research directions on roles of metals in PAH.

Evaluating the Feasibility of a Novel Firearm Injury Prevention Program for Pre-adolescent Children Through Health Care and Community-Based Partnerships: The Future Healers Program Pilot Study.

BACKGROUND: Firearm violence is an American public health crisis that negatively impacts children and disproportionately affects Black youth. Few firearm injury prevention programs have been described in pre-adolescent children. The Future Healers Program is a novel collaboration constructed via partnership between the medical school, trauma center, academic surgery department, and local non-profit community organization. Our study sought to evaluate if (1) partnering with community organizations facilitated recruitment of children with prior exposure to firearm violence and (2) the health care community was a potential trusted partner appropriate for program delivery. METHODS: Children aged 4-13 were recruited to join the program via news outlets and social media and in partnership with a local non-profit organization. Of the children and parents participating in the program, 48% (44/92) and 59% (38/64), respectively, completed an IRB-approved survey study. Pearson's chi-square, percentages, and 95% confidence intervals evaluated differences between children and caregivers on sociodemographic characteristics, firearm exposure (FE), firearm violence exposure (FVE), and perception of health care. Participant's residence was geocoded in relationship to incidents of firearm injury (2008-2021) in the same region. RESULTS: Caregivers (95%) and children (84%) reported substantial exposure to firearm violence and resided in areas with frequent firearm injury incidents. Notably, 82% of caregivers and 66% of children reported having a family member injured by gunfire. A high percentage of caregivers (79%) and children (91%) self-reported trust in the health care system. CONCLUSION: Partnerships between community organizations and health care systems can develop prevention programs that effectively recruit and engage pre-adolescent children impacted by firearm violence.

Exposure to Lead in Drinking Water Causes Cognitive Impairment via an Alzheimer's Disease Gene-Dependent Mechanism in Adult Mice.

Background: Exposure to lead (Pb) is a major public health problem that could occur through contaminated soil, air, food, or water, either during the course of everyday life, or while working in hazardous occupations. Although Pb has long been known as a neurodevelopmental toxicant in children, a recent and growing body of epidemiological research indicates that cumulative, low-level Pb exposure likely drives age-related neurologic dysfunction in adults. Environmental Pb exposure in adulthood has been linked to risk of late-onset Alzheimer's disease (AD) and dementia. Objective: Although the biological mechanism underlying this link is unknown, it has been proposed that Pb exposure may increase the risk of AD via altering the expression of AD-related genes and, possibly, by activating the molecular pathways underlying AD-related pathology. Methods: We investigated Pb exposure using a line of genetically modified mice with AD-causing knock-in mutations in the amyloid precursor protein and presenilin 1 (APPΔNL/ΔNL x PS1P264L/P264L) that had been crossed with Leprdb/db mice to impart vulnerability to vascular pathology. Results: Our data show that although Pb exposure in adult mice impairs cognitive function, this effect is not related to either an increase in amyloid pathology or to changes in the expression of common AD-related genes. Pb exposure also caused a significant increase in blood pressure, a well known effect of Pb. Interestingly, although the increase in blood pressure was unrelated to genotype, only mice that carried AD-related mutations developed cognitive dysfunction, in spite of showing no significant change in cerebrovascular pathology. Conclusions: These results raise the possibility that the increased risk of dementia associated with Pb exposure in adults may be tied to its subsequent interaction with either pre-existing or developing AD-related neuropathology.

Reorganization of pancreas circadian transcriptome with aging.

The evolutionarily conserved circadian system allows organisms to synchronize internal processes with 24-h cycling environmental timing cues, ensuring optimal adaptation. Like other organs, the pancreas function is under circadian control. Recent evidence suggests that aging by itself is associated with altered circadian homeostasis in different tissues which could affect the organ's resiliency to aging-related pathologies. Pancreas pathologies of either endocrine or exocrine components are age-related. Whether pancreas circadian transcriptome output is affected by age is still unknown. To address this, here we profiled the impact of age on the pancreatic transcriptome over a full circadian cycle and elucidated a circadian transcriptome reorganization of pancreas by aging. Our study highlights gain of rhythms in the extrinsic cellular pathways in the aged pancreas and extends a potential role to fibroblast-associated mechanisms.

Reorganization of Pancreas Circadian Transcriptome with Aging.

The evolutionarily conserved circadian system allows organisms to synchronize internal processes with 24-h cycling environmental timing cues, ensuring optimal adaptation. Like other organs, the pancreas function is under circadian control. Recent evidence suggests that aging by itself is associated with altered circadian homeostasis in different tissues which could affect the organ's resiliency to aging-related pathologies. Pancreas pathologies of either endocrine or exocrine components are age-related. Whether pancreas circadian transcriptome output is affected by age is still unknown. To address this, here we profiled the impact of age on the pancreatic transcriptome over a full circadian cycle and elucidated a circadian transcriptome reorganization of pancreas by aging. Our study highlights gain of rhythms in the extrinsic cellular pathways in the aged pancreas and extends a potential role to fibroblast-associated mechanisms.

The development of a derelict crab trap removal incentive program for commercial shrimpers.

Due to fishery-tailored gear, shrimpers are often affected by benthic marine debris, specifically derelict crab traps. To alleviate the impacts on the commercial shrimping industry in the Mississippi Sound, a team of natural resource professionals and stakeholders developed a derelict crab trap removal incentive program for commercial shrimpers. In three years, this program led to the removal of 2904 derelict crab traps from the north-central Gulf of Mexico at a total average cost of $35,595 per year to implement the program, or $53 per derelict crab trap. Results from this study showed the cost of the program could further be reduced while covering the same shrimping area, through the inclusion of fewer disposal locations and targeting active and engaged shrimpers. This program led to the removal of crab traps by non-registered shrimpers, indicating that the existence of the program and associated outreach could lead to improved environmental stewardship without an incentive.

The distribution and direct impacts of marine debris on the commercial shrimping industry.

Commercial shrimpers frequently encounter marine debris in their nets, resulting in economic impacts. Currently, no information existed on the spatial and temporal distribution of marine debris that shrimpers encounter and the subsequent economic impact on commercial shrimping. Twenty commercial shrimpers participated in a comprehensive data collection program (July 2020 through December 2020) within the north-central Gulf of Mexico, USA to characterize the quantity and impacts of marine debris. Derelict crab traps were an overwhelming issue for shrimpers. The type of fishing gear used influenced the type of marine debris encountered and the subsequent economic impacts. Surveyed shrimpers encountered marine debris on 19 % of tows and lost an average of 18.21 min, 7.88 kg of catch, and $6.37 in gear damage per tow with encounters, resulting in average annual losses of $6601 per shrimper. The results of this study show that marine debris encounters can have a large impact on the commercial shrimping industry.

Potential Roles of Metals in the Pathogenesis of Pulmonary and Systemic Hypertension.

Pulmonary and systemic hypertension (PH, SH) are characterized by vasoconstriction and vascular remodeling resulting in increased vascular resistance and pulmonary/aortic artery pressures. The chronic stress leads to inflammation, oxidative stress, and infiltration by immune cells. Roles of metals in these diseases, particularly PH are largely unknown. This review first discusses the pathophysiology of PH including vascular oxidative stress, inflammation, and remodeling in PH; mitochondrial dysfunction and metabolic changes in PH; ion channel and its alterations in the pathogenesis of PH as well as PH-associated right ventricular (RV) remodeling and dysfunctions. This review then summarizes metal general features and essentiality for the cardiovascular system and effects of metals on systemic blood pressure. Lastly, this review explores non-essential and essential metals and potential roles of their dyshomeostasis in PH and RV dysfunction. Although it remains early to conclude the role of metals in the pathogenesis of PH, emerging direct and indirect evidence implicates the possible contributions of metal-mediated toxicities in the development of PH. Future research should focus on comprehensive clinical metallomics study in PH patients; mechanistic evaluations to elucidate roles of various metals in PH animal models; and novel therapy clinical trials targeting metals. These important discoveries will significantly advance our understandings of this rare yet fatal disease, PH.

Patient-derived pancreatic cancer-on-a-chip recapitulates the tumor microenvironment.

The patient population suffering from pancreatic ductal adenocarcinoma (PDAC) presents, as a whole, with a high degree of molecular tumor heterogeneity. The heterogeneity of PDAC tumor composition has complicated treatment and stalled success in clinical trials. Current in vitro techniques insufficiently replicate the intricate stromal components of PDAC tumor microenvironments (TMEs) and fail to model a given tumor's unique genetic phenotype. The development of patient-derived organoids (PDOs) has opened the door for improved personalized medicine since PDOs are derived directly from patient tumors, thus preserving the tumors' unique behaviors and genetic phenotypes. This study developed a tumor-chip device engineered to mimic the PDAC TME by incorporating PDOs and stromal cells, specifically pancreatic stellate cells and macrophages. Establishing PDOs in a multicellular microfluidic chip device prolongs cellular function and longevity and successfully establishes a complex organotypic tumor environment that incorporates desmoplastic stroma and immune cells. When primary cancer cells in monoculture were subjected to stroma-depleting agents, there was no effect on cancer cell viability. However, targeting stroma in our tumor-chip model resulted in a significant increase in the chemotherapy effect on cancer cells, thus validating the use of this tumor-chip device for drug testing.

Differential Functional Responses of Neutrophil Subsets in Severe COVID-19 Patients.

Neutrophils play a significant role in determining disease severity following SARS-CoV-2 infection. Gene and protein expression defines several neutrophil clusters in COVID-19, including the emergence of low density neutrophils (LDN) that are associated with severe disease. The functional capabilities of these neutrophil clusters and correlation with gene and protein expression are unknown. To define host defense and immunosuppressive functions of normal density neutrophils (NDN) and LDN from COVID-19 patients, we recruited 64 patients with severe COVID-19 and 26 healthy donors (HD). Phagocytosis, respiratory burst activity, degranulation, neutrophil extracellular trap (NET) formation, and T-cell suppression in those neutrophil subsets were measured. NDN from severe/critical COVID-19 patients showed evidence of priming with enhanced phagocytosis, respiratory burst activity, and degranulation of secretory vesicles and gelatinase and specific granules, while NET formation was similar to HD NDN. COVID LDN response was impaired except for enhanced NET formation. A subset of COVID LDN with intermediate CD16 expression (CD16Int LDN) promoted T cell proliferation to a level similar to HD NDN, while COVID NDN and the CD16Hi LDN failed to stimulate T-cell activation. All 3 COVID-19 neutrophil populations suppressed stimulation of IFN-γ production, compared to HD NDN. We conclude that NDN and LDN from COVID-19 patients possess complementary functional capabilities that may act cooperatively to determine disease severity. We predict that global neutrophil responses that induce COVID-19 ARDS will vary depending on the proportion of neutrophil subsets.

Methodology and applicability of the human contact burn injury model: A systematic review.

The contact burn injury model is an experimental contact thermode-based physiological pain model primarily applied in research of drug efficacy in humans. The employment of the contact burn injury model across studies has been inconsistent regarding essential methodological variables, challenging the validity of the model. This systematic review analyzes methodologies, outcomes, and research applications of the contact burn injury model. Based on these results, we propose an improved contact burn injury testing paradigm. A literature search was conducted (15-JUL-2020) using PubMed, EMBASE, Web of Science, and Google Scholar. Sixty-four studies were included. The contact burn injury model induced consistent levels of primary and secondary hyperalgesia. However, the analyses revealed variations in the methodology of the contact burn injury heating paradigm and the post-burn application of test stimuli. The contact burn injury model had limited testing sensitivity in demonstrating analgesic efficacy. There was a weak correlation between experimental and clinical pain intensity variables. The data analysis was limited by the methodological heterogenicity of the different studies and a high risk of bias across the studies. In conclusion, although the contact burn injury model provides robust hyperalgesia, it has limited efficacy in testing analgesic drug response. Recommendations for future use of the model are being provided, but further research is needed to improve the sensitivity of the contact burn injury method. The protocol for this review has been published in PROSPERO (ID: CRD42019133734).

Neuropeptide Y tonically inhibits an NMDAR➔AC1➔TRPA1/TRPV1 mechanism of the affective dimension of chronic neuropathic pain.

Transection of the sural and common peroneal branches of the sciatic nerve produces cutaneous hypersensitivity at the tibial innervation territory of the mouse hindpaw that resolves within a few weeks. We report that interruption of endogenous neuropeptide Y (NPY) signaling during remission, with either conditional NPY knockdown in NPYtet/tet mice or intrathecal administration of the Y1 receptor antagonist BIBO3304, reinstated hypersensitivity. These data indicate that nerve injury establishes a long-lasting latent sensitization of spinal nociceptive neurons that is masked by spinal NPY-Y1 neurotransmission. To determine whether this mechanism extends beyond the sensory component of nociception, we used conditioned place aversion and preference assays to evaluate the affective component of pain. We found that BIBO3304 produced place aversion in mice when administered during remission. Furthermore, the analgesic drug gabapentin produced place preference after NPY knockdown in NPYtet/tet but not control mice. We then used pharmacological agents and deletion mutant mice to investigate the cellular mechanisms of neuropathic latent sensitization. BIBO3304-induced reinstatement of mechanical hypersensitivity and conditioned place aversion could be prevented with intrathecal administration of an N-methyl-d-aspartate receptor antagonist (MK-801) and was absent in adenylyl cyclase type 1 (AC1) deletion mutant mice. BIBO3304-induced reinstatement could also be prevented with intrathecal administration an AC1 inhibitor (NB001) or a TRPV1 channel blocker (AMG9801), but not vehicle. Intrathecal administration of a TRPA1 channel blocker (HC030031) prevented the reinstatement of neuropathic hypersensitivity produced either by BIBO3304, or by NPY knockdown in NPYtet/tet but not control mice. Our results confirm new mediators of latent sensitization: TRPA1 and TRPV1. We conclude that NPY acts at spinal Y1 to tonically inhibit a molecular NMDAR➔AC1 intracellular signaling pathway in the dorsal horn that is induced by peripheral nerve injury and drives both the sensory and affective components of chronic neuropathic pain.

Accumulation and distribution of marine debris on barrier islands across the northern Gulf of Mexico.

Marine debris is an economic, environmental, human health, and aesthetic problem posing a complex challenge to communities around the globe. To better document this problem in the Gulf of Mexico we monitored the occurrence and accumulation rate of marine debris at twelve sites on nine barrier islands from North Padre Island, Texas to Santa Rosa, Florida. With this information we are investigating three specific questions: (1) what are the major types/sources of marine debris; (2) does debris deposition have seasonal oscillations; and (3) how does debris deposition change spatially? Several trends emerged; plastic composed 69-95% of debris; there was a significant increase in debris accumulation during the spring and summer seasons; accumulation rates were ten times greater in Texas than the other Gulf States throughout the year; and the amount of debris accumulating along the shoreline could be predicted with high confidence in areas with high freshwater influx.

Abundance and characteristics of microplastics in beach sediments: Insights into microplastic accumulation in northern Gulf of Mexico estuaries.

Microplastics (plastic debris smaller than 5mm) represent a growing concern worldwide due to increasing amounts of discarded trash. We investigated microplastic debris on sandy shorelines at seven locations in a northern Gulf of Mexico estuary (Mobile Bay, AL) during the summer of 2014. Microplastics were ubiquitous throughout the area studied at concentrations 66-253× larger than reported for the open ocean. The polymers polypropylene and polyethylene were most abundant, with polystyrene, polyester and aliphatic polyamide also present but in lower quantities. There was a gradient in microplastic abundance, with locations more directly exposed to marine currents and tides having higher microplastic abundance and diversity, as well as a higher contribution by denser polymers (e.g. polyester). These results indicate that microplastic accumulation on shorelines in the northern Gulf of Mexico may be a serious concern, and suggest that exposure to inputs from the Gulf is an important determinant of microplastic abundance.

Lack of Impact of Posidonia oceanica Leaf Nutrient Enrichment on Sarpa salpa Herbivory: Additional Evidence for the Generalist Consumer Behavior of This Cornerstone Mediterranean Herbivore.

The fish Sarpa salpa (L.) is one of the main macroherbivores in the western Mediterranean. Through direct and indirect mechanisms, this herbivore can exert significant control on the structure and functional dynamics of seagrass beds and macroalgae. Past research has suggested nutritional quality of their diet influences S. salpa herbivory, with the fish feeding more intensively and exerting greater top down control on macrophytes with higher internal nutrient contents. However recent findings have questioned this notion and shown that herbivores do not preferentially feed on macrophytes with higher nutrient contents, but rather feed on a wide variety of them with no apparent selectivity. To contribute to this debate, we conducted a field fertilization experiment where we enriched leaves of the seagrass Posidonia oceanica, a staple diet for S. salpa, and examined the response by the herbivore. These responses included quantification of leaf consumption in fertilized and non-fertilized/control plots within the bed, and food choice assays where fertilized and non-fertilized/control leaves were simultaneously offered to the herbivore. Despite the duration of leaf exposure to herbivores (30 days) and abundant schools of S. salpa observed around the plots, leaf consumption was generally low in the plots examined. Consumption was not higher on fertilized than on non-fertilized leaves. Food choice experiments did not show strong evidence for selectivity of enriched leaves. These results add to a recent body of work reporting a broad generalist feeding behavior by S. salpa with no clear selectivity for seagrass with higher nutrient content. In concert, this and other studies suggest S. salpa is often generalist consumers not only dictated by diet nutrient content but by complex interactions between other traits of nutritional quality, habitat heterogeneity within their ample foraging area, and responses to predation risk.